Enteroviruses cause millions of infections each year around the world and the US. These RNA viruses include more than 100 serotypes that are grouped into 4 species (A, B, C and D). Enterovirus A71 (EV71) is a specific strain that is known to be associated with respiratory, hand, foot, and mouth disease. The neurologic complications of EV71 infection, which include brain-stem encephalitis, acute flaccid paralysis, and aseptic meningitis, are of greater clinical significance and can occur in the absence of the more commonly encountered cutaneous manifestations. Over the past decade, there have been more than 6 million cases of EV71 infection worldwide, and more than 2000 of them have been fatal. The infection is not exclusive to Asia. In 2007, there was an EV71 outbreak in The Netherlands and transmission from Japan to the EU and then onward to Canada was also documented (2009 and 2014). An inactivated vaccine has been recently developed but given that EV71 is transmitted by the oral/fecal route, mucosal immunity may be required to prevent transmission, which otherwise would maintain the risk to infection and disease, and the need for continued high coverage vaccination. The central goal of this project is to identify determinants of attenuation using a novel highly accurate genetic approach developed in our lab, with the long-term goal of developing live-attenuated, safe, and effective EV71 vaccine. The theoretical framework of this application is that genetic variation is central to virus adaptation within the host and invasion of tissues where the virus causes disease. Accordingly, limiting the ability of the viruses to adapt to new tissues will prevent intra-host spread and invasion of the central nervous system. As proof-of-concept, we have identified in poliovirus, and now in EV71, mutations within the virus polymerase that reduce genetic diversity by increasing replication fidelity (high fidelity or HiFi) and reducing RNA recombination rates (RecD). These variants are nevertheless highly immunogenic, and the poliovirus strains developed in our lab are currently in clinical trial. Using the experience in poliovirus, we now propose to isolate, characterize and test in mice the efficacy and safety of adaptation-defective EV71 variants as potential live- attenuated EV71 vaccine candidates.